The metabolic strategies used by bacteria to cope with life in the absence of oxygen have been primarily studied in strict anaerobes. Little effort has been made to approach this problem in facultative anaerobes like enterobacteria (e.g., E. coli and Salmonella) for which genetic methods have been developed. Most work on the biochemistry and genetics of enterobacteria has been done using cells growing under aerobic conditions. We propose to investigate the anaerobic metabolism of the facultative anaerobe Salmonella typhimurium. Vitamin B12 (cobalamin) is a essential nutrient for many forms of life (excluding plants) including man. The biosynthesis of this important macromolecule is not well understood, and practically nothing is known about the environmental factors, and the mechanisms that regulate the synthesis of B12 at the molecular level. Salmonella makes B12 de novo but only during anaerobic growth conditions. The use of sophisticated genetics in this bacterium has yielded important information about the genomic organization and transcriptional regulation of the cobalamin biosynthetic (cob) genes. We will initiate a comprehensive approach to the characterization of the B12 biosynthetic enzymes. For this purpose, we will use modern chemical, biochemical, molecular biological, and genetic techniques. Genetic and molecular biological experiments will be performed to determine the number of gene products involved in the biosynthesis of the lower ligand of B12 (i.e., dimethylbenzimidazole, DMB), and in the synthesis of the nucleotide loop, which links DMB to the rest of the molecule. We will use biochemical and genetic techniques to establish the metabolic precursors of DMB, and its biosynthetic route(s). We have isolated a large number of mutants that will help us identify enzymic as well as regulatory functions of this pathway. Assays for a number of proposed enzymes will be set up, and used in their purification in order to understand the underlying chemical events leading to the synthesis of this macromolecule. We plan to study the role of newly discovered cob genes that map outside the reported cob operons, and which have different phenotypes in response to oxygen. These genes may be involved in interactions between the biosynthetic pathway and the sophisticated transport systems for this vitamin. We propose to continue investigating B12 biosynthesis in hopes of increasing our understanding of this vitamin and the anaerobic lifestyle of Salmonella.